Industrial biocatalysis demands the set-up of bioprocesses with high substrate loadings, leading to high productivities in competitive reaction times. In this respect, the use of strong electrophilic reagents such as short-chain and highly reactive aldehydes typically leads to enzyme deactivation, thus decreasing the biocatalytic efficiency far from industrial requests.
In the realm of biocatalyis, C—C bond forming reactions represent a core strategy from which many valuable (optically active) building blocks and pharmaceutical precursors can be furnished. In this area, for example, hydroxyketoacids can be biocatalytically prepared by aldol addition of pyruvate to an aldehyde by a pyruvate-dependent aldolase. The hydroxyketoacid moiety can be found in a plethora of natural occurring important products such as sialic acids typically found in mammalian and bacterial glycoconjugates. A number of sialic acid derivatives have been prepared using biocatalytic approaches involving pyruvate-dependent aldolases catalyzing the aldol addition of pyruvate to analogues and derivatives of N-acetyl-mannosamine. In this area, class I pyruvate-dependent aldolases are normally used with rather strict selectivity for the donor substrate (i.e. pyruvate) but with a fairly broad tolerance of acceptor substrates such as a number of sugars and their derivatives larger or equal to pentoses. However, small open-chain aldehydes such as glyceraldehyde, glycolaldehyde, aliphatic, aromatic aldehyde are not acceptable. Likewise, the inherent high reactivity of those aldehydes tends to deactivate the enzymes (especially when used at high substrate loadings), making the processes often inefficient from an economic viewpoint.
Given the potential of lyases and C—C bond forming reactions for industrial purposes, and the aforementioned challenges found in the prior art, what is needed is the set-up of biocatalytic processes comprising enzymes that may be largely stable and active under very high substrate loadings, leading to robust industrial bioprocesses.